Emerging Antiviral Resistance in Congenital Cytomegalovirus Infection In the United States, cytomegalovirus (CMV) is the leading cause of congenital infection and leads to permanent sequelae in over 6,000 infants per year. Potential benefits for hearing and neurodevelopmental outcomes have been demonstrated in affected infants with central nervous system symptoms after a 6-week course of ganciclovir. Subsequent studies are evaluating 6 weeks versus 6 months of oral valganciclovir to determine if a longer duration of antiviral therapy will further improve outcomes. Our preliminary studies indicate that the population of congenitally infected infants could be at high risk for developing antiviral resistance while on therapy. Our proposed investigation into the emergence of antiviral resistance in infants with congenital CMV disease seeks to address a critical gap in medical knowledge of optimal therapeutic management in this vulnerable population. We hypothesize that congenitally infected infants are at significant risk for developing antiviral resistance and that such resistance will worsen the hearing and neurodevelopmental outcomes of affected infants. An applied research strategy will be employed to test this hypothesis in an effort to contribute to our knowledge of how to most effectively improve long-term outcomes. In a controlled study population of over 100 infants with congenital CMV infection who are exposed to prolonged antiviral therapy, both conventional and next-generation DNA sequencing techniques will be utilized to demonstrate the emergence of majority and minority subpopulations of drug-resistant CMV strains. Next- generation sequencing will be further applied to investigate the mixed virus population dynamics of CMV infections with and without selective antiviral pressure. Recombinant phenotyping of engineered viruses containing novel UL97 and UL54 sequence variants will explore previously unrecognized resistance mutations. Confirmation of new resistance mutations is an important aspect of advancing the field of knowledge of CMV antiviral resistance and is directly applicable beyond our study population. The candidate Principal Investigator for these studies is a junior faculty member in the Division of Pediatric Infectious Diseases at the University of Alabama at Birmingham, with a training background and research environment that provides excellent support for the accomplishment of the study aims. Short-term training objectives are laid out in a multifaceted career development plan that integrates a combination of didactic and hands-on training that will ensure that the candidate matures as a researcher during the implementation of these studies. Career development over the course of the award period ultimately will aim to prepare the candidate for a competitive R01 submission in keeping with his long-term career goal of becoming an independent investigator. RELEVANCE: Cytomegalovirus (CMV) is the most common congenital infection in the United States, often leading to permanent disability in the form of hearing loss and/or neurodevelopmental delay. Antiviral therapy improves clinical outcomes, but the clinical significance of antiviral resistance is unknown in this population. This project investigates the emergence of antiviral resistance in infants with symptomatic congenital CMV infection, explores the impact of developing resistance on clinical outcomes, and uses new technologies to advance the field of CMV resistance analysis.